The enzyme ribonucleotide reductase is responsible for the synthesis of the deoxyribonucleotide precursors of DNA and thus plays a central role in cell proliferation. The level of ribonucleotide reductase activity undergoes large changes in the course of the cell cycle, and there is some evidence for both a functional and physical relationship between this enzyme and other enzymes required for DNA replication. This relationship will be examined using mouse L cells synchronized at various stages in the cell cycle. In order not to disturb the physiological macromolecular environment of ribonucleotide reductase, these studies will be carried out in intact cells that have been selectively permeabilized to small molecules by treatment with dextran sulfate or digitonin. The proposed research will involve the study of possible changes in the properties of ribonucleotide reductase during the transitions from G1 to S phase and from M to G1 phase, including relative activity with the four ribonucleotide substrates, hydrogen donor specificity, and intracellular localization, as well as the amounts of the two subunits measured immunologically. It also includes the examination of the functional relationship between ribonucleotide reductase and DNA replication in terms of possible channeling of ribonucleotides into DNA, mediated by a multienzyme complex. Finally, attempts will be made to obtain direct evidence for a multienzyme replication complex involving ribonucleotide reductase by in vivo cross-linking experiments.